Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Abstract Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of α-helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the obtained protein was predicted to be allergenically active. Moreover, we report on the possible role of the Sol g 4.1 venom protein, which significantly reduced the PD50 from 0.027 to 0.013% in paralyzed crickets via synergistic effects after interactions with piperidine alkaloids. Conclusions: The primary structure of Sol g 4.1 showed high similarity to that of venom proteins in the Solenopsis 2 and 4 family. Those proteins are life-threatening and produce IgE-mediated anaphylactic reactions in allergic individuals. The possible function of this protein is the binding of the interior hydrophobic pockets with piperidine alkaloids, as determined by the analysis of the structural model and PD50 test.

摘要 背景：火蚁毒液是由碱性哌啶生物碱（piperidine alkaloids）、多种生物活性肽及蛋白质组分构成的复杂混合物，其中包含多种主要致敏蛋白。热带火蚁（Solenopsis geminata）是一类重要的螫刺蚁类，其叮咬可引发过敏反应（anaphylaxis）及严重医学病症。尽管蚁类毒液独有的致敏蛋白（尤其是索伦蚁属2型及4型（Solenopsis 2 and 4））的生物学活性仍未明确，但现有研究认为这类蛋白在介导毒液中哌啶衍生物的毒性效应中发挥关键作用。
方法：本研究针对Sol g 4.1毒液蛋白的cDNA克隆、测序及三维结构展开解析。本研究在大肠杆菌（E. coli）中重组表达了Sol g 4.1蛋白（rSol g 4.1），并通过以哌啶半数有效剂量（PD50）麻痹蟋蟀的实验，表征了其作为疏水结合蛋白的潜在功能。此外，本研究通过小鼠制备抗血清以探究Sol g 4.1的致敏特性，经蛋白质免疫印迹（Western blotting）验证，该抗血清可特异性结合Sol g 4.1蛋白。
结果：经十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）检测，Sol g 4.1蛋白的分子量为16 kDa。该蛋白的完整cDNA序列长度为414 bp，包含一段由19个氨基酸残基组成的前导序列。基于预测的三维结构模型，该蛋白包含6个半胱氨酸残基，推测可形成3对二硫键，进而构成内部疏水口袋并维持蛋白结构稳定。经SDS-PAGE检测，重组Sol g 4.1蛋白以包涵体形式表达。本研究采用透析法对重组蛋白进行复性，使其恢复天然构象。通过圆二色谱（circular dichroism）分析证实，该蛋白的二级结构以α螺旋为主，同时验证了此前预测的三维结构模型。小鼠致敏分析结果显示，该重组蛋白具备致敏活性。此外，本研究揭示了Sol g 4.1毒液蛋白的潜在功能：该蛋白可与哌啶生物碱产生协同效应，使麻痹蟋蟀的PD50从0.027%显著降至0.013%。
结论：Sol g 4.1的一级结构与索伦蚁属2型及4型家族的毒液蛋白具有高度同源性。这类蛋白可危及生命，并可在过敏个体中引发免疫球蛋白E（IgE）介导的过敏反应。结合结构模型分析与PD50实验结果，该蛋白的潜在功能为通过内部疏水口袋与哌啶生物碱结合。